The influenza virus is humanity's most prolific viral assassin, killing tens of thousands of Americans in a typical year and as many as 20-100 million people worldwide when new a pandemic strain emerges. In order to be infectious, an individual virion must incorporate (package) at least one copy of each of the eight linear RNA segments that make up its genome. Pandemics are thought to arise when an existing strain packages one or more segments from another strain in a dually-infected host, yielding a genetic reassortant with novel immunologic and pathogenic properties. Understanding influenza RNA packaging is therefore critical for understanding and, perhaps, controlling, this lethal re-emerging virus. To date, however, the cis-acting determinants required for packaging (i.e., the packaging signals) have not been fully elucidated for any segment, and the trans-acting factors involved are unknown. Our laboratory has extensive experience in defining the requirements for RNA packaging by HIV. In recent studies, we have localized the signals required for efficient packaging to within discrete 45- to 200-base regions at one or both ends of three influenza RNA segments. We now propose to map the active regions in detail and to determine which specific features of RNA sequence and/or secondary structure are required for packaging of these and the other five segments. We will also pursue our surprising observation that 3'or 5'packaging regions are not simply interchangeable among segments, which implies that as-yet-unknown combinatorial rules govern their interactions in packaging. In addition, we have obtained compelling evidence implicating the influenza PA protein (a component of the trimeric viral RNA polymerase) as a trans-acting factor required for packaging all eight viral RNA segments. We will now investigate the molecular pathway by which PA and other viral proteins recognize the packaging signals in viral RNA segments and directing their incorporation into virions. The studies we propose will help elucidate the linkage between packaging and virion assembly, will extend our understanding of influenza genetics and reassortment, and may reveal vulnerable new targets for antiviral drug development.